A major achievement in animal cell technology consists in the establishment of optimal conditions for cryopreservation of living cells and tissues, since long-term storage of valuable materials is enhanced. With cryopreservation, the intrinsic technical or practical difficulties associated with handling biomaterials such as (i) the limited shelf-life of isolated tissues, and (ii) the limited availability of tissues as source of cells for research and clinical applications, are reduced or avoided. From this point of view, development of preservation protocols for living cells and tissues has acquired remarkable importance for tissues susceptible for transplantation.
After early work on cryopreservation, that reported the successful use of glycerol to prevent damage of cells during freezing (Polge et al, 1949, Nature 164:666), several strategies to preserve cell and tissue structure, viability and metabolism have been attempted. The methods of preserving cells and tissues include: special isotonic buffered solutions; specific thawing schedules; the use of cryoprotectant agents; and approaches that comprise slow, rapid or ultrarapid freezing; all in order to prevent the otherwise inevitable destruction of living samples by handling. In general, when freezing methods are used, a major factor responsible for tissue damage is phase change (liquid to crystalline solid water), since ice formation is accompanied by changes in electrolyte concentration and pH, dehydration, and other factors not understood. These deleterious effects have been reduced by addition of cryoprotective agents and by carefully controlled freezing protocols.
Cryoprotective agents fall into two categories. One category acts by permeating the cell membrane and reducing the intracellular water concentration (e.g. glycerol, dimethyl sulfoxide (DMSO), and monosaccharides such as mannose, xylose, glucose, ribose and fructose). The other is non-permeating agents, the mechanism of action of which is not clear. Commonly employed non-permeating cryoprotectants include polyvinyl-pyrrolidone (PVP), hydroxyethyl starch (HES), disaccharides (such as sucrose), and sugar alcohols (polyalcohols such as mannitol). Recently, freezing protocols which combine both permeating and non-permeating agents have been developed.
Freezing of isolated cells has become routine with glycerol and DMSO being the predominate cryoprotectants (Coriell, L. L., 1979, Methods in Enzymology LVIII, pages 29-36; Doyle et al, 1994, Cell & Tissue Culture: Laboratory Procedures. J. Wiley & Sons, pages 4C: 1. 1-4C:2.4). Animal embryos also have been successfully preserved in the presence of glycerol (Whittingham et al, 1972, Science 178:411-414; Niemann et al, 1993, Mol. Reprod. Develop. 36:232-235). Drying and freeze-drying, on the other hand, have given excellent results only when used to preserve biomaterials other than living mammalian cells or tissues. For example, freeze-drying is now extensively used for proteins, protein mixtures and bacteria; however, the first attempts for lyophilization of mammalian cells and tissues were unsuccessful (Greaves, R. I. N., 1960, Ann. N.Y. Acad. Sci. 85:723-728). The recent discovery of biochemical adaptations of living organisms to survive complete dehydration (anhydrobiosis) (Crowe & Madin, 1975, J. Exp. Zool. 193:323-334; Womersley & Smith, 1981, Comp. Biochem. Physiol. 70B:579-586; reviewed by Womersley, 1981, Comp. Biochem. Physiol. 70B:679-678) or freezing (Constanzo et al, 1993, J. Exp. Zool. 181:245-255; King et al, 1993, Am. J. Physiol. 265:R1036-RI042; Karow et al, 1991, BioScience 41:155-160; Storey, 1990, Am. J. Physiol. 258:R559-R568), suggested new methods for preservation of mammalian cells or tissues.
It is believed that carbohydrates (such as trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, among others) and polyols (such as sorbitol and myo-inositol) might confer dehydration protection (Womersley & Smith, supra) and freeze-tolerance (Storey & Storey, 1988, Physiol. Rev. 68:27-84), in part through water replacement around cell membranes (Crowe et al, 1984, Arch. Biochem. Biophys. 232:400-407; Crowe et al, 1984, Biochem. Biophys. Acta 769:141-150). One of the most interesting carbohydrates that might be involved in dehydration-resistance and freeze-tolerance is glucose. This monosaccharide, possessive of extremely important functions in the metabolism of vertebrate cells, appears to have an important role in freeze-tolerance in a variety of frog species (King et al, 1993, supra; Constanzo et al, 1993, supra; Storey & Storey, 1988, supra). Data obtained from analysis of freeze-tolerant species suggest that during freezing of those animals (i) blood concentration of glucose increases significantly; (ii) glucose might be as an energy source in the anoxic and ischemic state imposed by the freezing; and (iii) glucose might function as a metabolic depressant (Storey & Storey, 1988, supra).
Sucrose has been used as a component in cryopreservation solutions used for corneal tissue storage (Madden et al, 1993, Cryobiology 30:135-157; Rich & Armitage, 1991, Cryobiology 28:159-170; McCarey et al, 1973, Cryobiology 10:298-307) and embryonic tissue freezing (Isachenko et al, 1993, Cryobiology 30:432-437). Also glucose and other carbohydrates have been used for red blood cell lyophilization (Goodrich et al., U.S. Pat. Nos. 4,874,690; 5,171,661 and 5,178,884). However, red blood cell preservation does not require maintenance of tissue-type structural organization, which would be required for tissue transplantation. In the above methods, red blood cell viability has been determined only by quantitation of erythrocyte lysis, hemoglobin recovery, or assay of glycolytic enzymes (see Goodrich et al, U.S. Pat. Nos. 4,874,690 and 5,178,884), but not by parameters related to cell integrity and tissue organization, such as protein synthesis and secretion.
Green and collaborators described a method for culturing human epidermal keratinocytes (Rheinwald & Green, 1975, Cell 6:331-343), that has been extended to other cultured epithelial cells. Under such culture conditions, stratified epithelial sheets suitable for transplantation onto large burn surfaces, ulcerations and other skin wounds are obtained (Gallico et al, 1984, New Eng. J. Med. 311:448-451; Heighten et al, 1986, J. Am. Acad, Dermatol. 14:399-405). The cultured epithelia obtained through this procedure have also been used as allografts for temporary wound dressing (T. J. Phillips et al., 1989, J. Am. Acad. Derm. 21:191; Bolivar-Flores et al, 1990, Bums 16:3-8). Epithelial cell cultures have become a powerful tool for body surface reconstruction, however, their limited shelf-life has restricted their use to those medical facilities that are not too far away from the production facility. After dispase detachment of epithelial sheets for transportation to the hospital, shelf-life is short. Therefore, the establishment of a preservation method for the cultured sheets should permit their banking and also, their shipment worldwide. In this regard, some strategies have been attempted. Several authors have developed cryopreservation methods based on the use of glycerol or dimethyl sulfoxide as cryoprotectants, following a specific freezing protocol (see Cancedda and De Luca, 1994, U.S. Pat. No. 5,298,417). Others have cryopreserved cultured epithelial sheets with media containing both cell-penetrating glass-forming agents (specifically glycerol) and non-penetrating protectant agents (preferably polyvinylpyrrolidone (PVP), dextran or hydroxyethyl starch) (see Tubo et al, 1992, U.S. Pat. No. 5,145,770). However, these methods require a specific and elaborate freezing protocol, and a thawing protocol that appears to impose difficulties in the wide use of these tissues in the clinical field; or a possible impairment of the medical efficacy due to difficulties in the thawing and rinsing protocols required in order to use such epithelial sheets.